Transplantation Tolerance in NF-B-Impaired Mice Is Not Due to Regulation but Is Prevented by Transgenic Expression of Bcl-xL
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免疫学杂志 2005年第6期
Abstract
NF-B is a key regulator of transcription after TCR and costimulatory receptor ligation. To determine the role of T cell-intrinsic NF-B activation in acute allograft rejection, we used IBN-Tg mice (H-2b) that express an inhibitor of NF-B restricted to the T cell compartment. We have previously shown that these mice permanently accept fully allogeneic (H-2d) cardiac grafts and secondary donor skin grafts, and that splenocytes from these tolerant mice have reduced alloreactivity when restimulated in vitro. These results were compatible with either deletion or suppression of allospecific T cells as possible mechanisms of tolerance. The aim of this study was to investigate the mechanism of transplant tolerance in these mice. IBN-Tg mice did not have increased numbers or function of CD4+CD25+ regulatory T cells either before or after cardiac transplantation. In addition, tolerance could not be transferred to fresh NF-B-competent T cells and was not permissive for linked suppression to skin grafts sharing donor and third-party alloantigens, suggesting that dominant suppression is not the mechanism by which IBN-Tg mice achieve tolerance. In contrast, overexpression of the antiapoptotic protein Bcl-xL in T cells from IBN-Tg mice resulted in effective rejection of cardiac allografts and correlated with an increased frequency of splenocytes producing IFN- in response to alloantigen. Together, these results suggest that the death of alloreactive T cells may be partly responsible for the transplantation tolerance observed in mice with defective T cell-intrinsic NF-B activation.
Introduction
Solid organ transplantation is often the only cure for end-stage organ failure. However, unless tissues are donated by identical twins, transplantation is limited by the occurrence of acute allograft rejection. Acute allograft rejection is mediated by T lymphocytes, and its prevention requires transplant recipients to take life-long immunosuppressive therapies, with the potential complications of infections and virus-induced tumors that are common with chronic immunosuppression. Therefore, the goal of transplantation research is to develop immunosuppressive regimens that inactivate only alloreactive T cells, leaving the rest of the immune system competent to react against pathogens and tumor Ags, a state termed donor-specific tolerance. Thus, understanding the biochemical pathways in T cells that are necessary to mount acute allograft rejection episodes is essential for the design of such novel therapies.
T cell activation follows engagement by the TCR of specific peptide/MHC complexes displayed on the surface of APCs, concurrently to the ligation of coreceptors (CD4 or CD8) and costimulatory receptors on T cells. An essential costimulatory receptor for naive T cells is CD28, which binds B7-1 (CD80) and B7-2 (CD86) on APCs (1). Engagement of TCR in the absence of CD28 ligation results in T cell death or T cell inactivation (2, 3). Biochemical signals transduced upon TCR/CD28 engagement include activation of the Src kinase Lck, phosphorylation of ITAM motifs in TCR -chains, and recruitment and activation of the tyrosine kinase ZAP70 and subsequently of the scaffolding adaptors Lat and SLP76 that are essential for the formation of signalosomes localized to lipid rafts at the plasma membrane (4). These signalosomes promote activation of downstream transcription factors that in naive T cells include NFAT, AP-1, and NF of the B cell -chain (NF-B). Although these events have been well established in vitro, it is not clear whether all these pathways take place after T cell activation in vivo or what steps are necessary for mounting a productive immune response in the context of transplant rejection. Several groups have begun investigating the role of transcription factors during acute allograft rejection. For instance, inhibition of NFAT using cell-permeable inhibitory peptides resulted in prolongation of islet allograft survival in mice (5).
Our laboratory has chosen to concentrate on the role of NF-B activation in T cells during acute allograft rejection, because NF-B had been implicated in cell survival, proliferation, and cytokine production in several model systems in vitro (6) and therefore was likely to play an important role in immune responses in vivo. The Rel/NF-B family of proteins is composed of five members that each contain a Rel homology domain (RHD)5 important for protein and DNA binding. p50 and p52 bind DNA well, but have weak transactivating capacity, whereas RelA (p65), RelB, and c-Rel are poor DNA binders, but contain a DNA transactivating domain. Typical NF-B molecules are composed of a heterodimeric combination of p50 or p52, bound with p65, RelB, or c-Rel. T cells contain all NF-B family subunits. In resting cells, NF-B dimers are sequestered in the cytoplasm by IB family members. IB molecules bind to and cover the nuclear localization signals on NF-B heterodimers, thus preventing their translocation to the nucleus (6). Engagement of TCR and CD28 results in activation of IB kinases (IKK), which phosphorylate IB. The phosphorylated IB is then ubiquitinated and subsequently degraded by the proteasome. This exposes the nuclear localization signal on the NF-B dimer, which can move into the nucleus and activate gene transcription. Nuclear translocation of NF-B subunits has been reported in cardiac allografts at the time of T cell infiltration and initiation of rejection (7).
Many immunosuppressive drugs commonly used to prevent or treat allograft rejection inhibit NF-B as part of their mechanism of action, although they may also exert many other effects. Corticosteroids, for instance, induce de novo transcription of IB molecules and therefore inhibit further NF-B activation (8, 9). Several nonsteroidal anti-inflammatory drugs reduce the phosphorylation of IB by the IKK complex (10). Even cyclosporin A, a compound known for its inhibitory activity of NFAT cells, has been shown to reduce proteasomal degradation of phosphorylated IBs (10). The immunosuppressive activity of these drugs is probably due at least in part to their NF-B inhibitory effects.
Transplantation experiments have been performed on mice genetically deficient in single NF-B subunits, but prolongation of graft survival in these mice has been slight, presumably due to redundancy and compensation by other subunits. Thus, p50- or p52-deficient mice effectively reject cardiac allografts (11, 12). The role of T cell-intrinsic NF-B activation during allograft rejection has been reinvestigated using mice that have reduced NF-B activity selectively in T cells. These mice express a super-repressor transgenic form of IB driven by the Lck proximal promoter and CD2 locus control region (13). This transgene bears a deletion of the region containing the serines that serve as targets for the IKK complex. Thus, this mutant IB cannot be phosphorylated by the IKK complex and therefore retains the NF-B subunits to which it is bound in the cytoplasm. We and others have shown that the majority of IBN-Tg mice permanently accept heart transplants (14, 15), but reject skin allografts (15). However, IBN-Tg mice tolerant to cardiac allografts permanently accept secondary donor, but not third-party, skin allografts, indicative of donor-specific tolerance (15). Splenocytes from these mice had reduced reactivity against donor, but not third-party, Ags (15), suggesting that alloreactive T cells had been deleted or were hyporesponsive either because they had become anergic or because they were being suppressed by regulatory cells. Several transplantation models in which long-term graft survival has been achieved have identified either T cell apoptosis or T cell regulation as a major mechanism to attenuate rejection (16). In this study we have investigated whether graft acceptance in IBN-Tg mice is due to regulation or deletion of alloreactive T cells. Tolerance in this model was not transferable to fresh wild-type T cells. In contrast, overexpression in T cells of the antiapoptotic protein Bcl-xL resulted in cardiac allograft rejection by IBN-Tg mice, suggesting that apoptosis, but not regulation, may be the main mechanistic pathway of tolerance in these mice.
Materials and Methods
Mice
IBN-Tg mice (H-2b) have been previously described (13) and were a gift from M. Boothby (Vanderbilt University, Nashville, TE). Bcl-xL-Tg mice (H-2b) have been previously described (17) and were provided by C. Thompson (University of Pennsylvania, Philadelphia, PA). IBN-Tg and Bcl-xL-Tg mice were intercrossed to generate double-transgenic mice (Bcl-xL/IBN-Tg). C57BL/6 (B6; H-2b) and BALB/c (H-2d) were purchased from Frederick Cancer Research Facilities. B6/RAG1-deficient (RAG1-KO; H-2b) mice were bred in our animal facility. Animals were housed in ventilated racks in a specific pathogen-free animal facility. Experiments were performed in agreement with our Institutional animal care and use committee and according to the National Institutes of Health guidelines for animal use.
Heart and skin transplantation
Abdominal heterotopic cardiac transplantation was performed using a technique adapted from that originally described by Corry et al. (18). Cardiac allografts were transplanted in the abdominal cavity by anastomosing the aorta and pulmonary artery of the graft end-to-side to the recipient’s aorta and vena cava, respectively. The day of rejection was defined as the last day of a detectable heartbeat in the graft. Graft rejection was verified in selected cases by necropsy and pathological examination of H&E-stained graft sections.
Cervical cardiac transplantation was performed using a technique adapted from Chen (19). The cardiac graft was placed under the skin of the front of the neck. The innominate artery of the donor heart was anastomosed end-to-end to the right common carotid artery of the recipient. The pulmonary artery of the donor heart was anastomosed end-to-end to the external jugular vein of the recipient.
Some B6 mice were transplanted with BALB/c hearts and treated with a combination of anti-CD40L (MR1, 1 mg i.p. on days 0, 7, and 14 posttransplantation; Ligocyte Pharmaceuticals) and donor-specific transfusion (DST; 5 x 106 BALB/c splenocytes i.v. on day 0).
Flow cytometry
Splenocytes were isolated and processed into single cell suspensions. Cells were stained with anti-CD4-FITC- and anti-CD25-PE-coupled Abs (BD Pharmingen) and analyzed by flow cytometry (FACScan I or II; BD Biosciences).
Functional suppressor capacity of regulatory T cells (Tregs)
To assess the regulatory capacity of CD4+CD25+ T cells, splenocytes from unmanipulated B6 and IBN-Tg mice either unmanipulated or transplanted >50 days previously with a BALB/c heart were isolated and stained with dialyzed anti-CD4-FITC and anti-CD25-PE Abs. Cell populations were sorted using a MOFLO-HTS cell sorter (DakoCytomation) into CD4+CD25– (responding cells) or CD4+CD25+ (regulatory cells). Responding cells were resuspended in DMEM supplemented with 10% FBS, penicillin (100 U/ml), streptomycin (100 μg/ml), HEPES, 2-ME (50 μM), and additional amino acids and seeded in 96-well, round-bottom plates (3 x 104 cells/well) in the presence of soluble anti-CD3 (10 μg/ml) and irradiated (2000 rad) syngeneic splenocytes (15 x 104 cells/well). Increasing numbers of CD4+CD25+ T cells were added to the wells. Supernatants were collected at 24 h, and the concentration of IL-2 was measured by ELISA using Ab pairs (BD Pharmingen). Parallel plates were pulsed with [3H]thymidine for the last 8 h of a 72-h culture (1 μCi/well).
Adoptive transfer of T cells
Spleens were harvested from unmanipulated B6 mice or anti-CD40L- plus DST-treated, long-term BALB/c cardiac graft-accepting B6 mice or IBN-Tg mice that had accepted a BALB/c heart transplanted >50 days previously. Indicated numbers of splenocytes were resuspended in 150 μl of PBS and injected i.v. into the retro-orbital plexus of recipient mice. Recipient mice in some experiments were syngeneic RAG1-KO mice that had been transplanted 1 day previously with BALB/c cardiac allografts. In other experiments, recipient mice were anti-CD40L- plus DST-treated, long-term BALB/c cardiac graft-accepting B6 mice or IBN-Tg mice that had accepted a cervical BALB/c heart for >50 days. These recipients received a second abdominal BALB/c cardiac transplant 1 day before the adoptive transfer.
IFN- ELISPOTs
Splenocytes from untransplanted mice or from mice transplanted 25 days previously with a BALB/c heart (106/well) were restimulated in vitro with irradiated B6 or BALB/c splenocytes (4 x 105/well) and incubated for 24 h in a 5% CO2 incubator. The ELISPOT assay was conducted according to the instructions of the manufacturer (BD Biosciences), and the numbers of spots per well were calculated using the ImmunoSpot Analyzer (CTL Analyzers LLC).
Results
IBN-Tg mice do not have increased numbers of CD4+CD25+ regulatory T cells either before or after heart allograft acceptance
CD4+CD25+ T cells are naturally occurring regulatory cells that develop in the thymus. It was possible that reduced NF-B activation during thymic maturation promoted the development of Treg at the expense of effector T cells, so that the ratio of CD4+CD25+ to CD4+CD25– T cells be altered and favor tolerance induction. To test this hypothesis, we compared the number of CD4+CD25+ T cells in wild-type and IBN-Tg littermates. Consistent with the known reduction in the number of total CD4+ T cells in IBN-Tg mice compared with wild-type mice, the total number of CD4+CD25+ was also reduced in the spleens of these mice (Table I). However, the percentage of CD4+CD25+ within the CD4+ population was similar to that in wild-type mice in the spleen of both unmanipulated (Table I) and tolerant (data not shown) IBN-Tg mice. Similar results were obtained in lymph nodes of these animals (data not shown). This result indicates that reduced NF-B activation in T cells does not favor the development of a greater proportion of CD4+CD25+ T cells either spontaneously or after alloantigenic encounter.
Table I. IBN-Tg mice do not have an increased number of CD4+CD25+ cellsa
T cells from both unmanipulated and tolerant IBN-Tg mice do not have an intrinsically better capacity to regulate or be regulated than wild-type T cells
Although the number of CD4+CD25+ T cells was not increased in IBN-Tg mice when compared with wild-type mice, it was possible that IBN-Tg CD4+CD25+ T cells had increased suppressor function on a per cell basis or that IBN-Tg CD4+CD25– cells were more susceptible to suppression than wild-type T cells. To explore these possibilities, CD4+ cells from unmanipulated IBN-Tg and control littermates mice were sorted into populations of CD4+CD25+ and CD4+CD25– cells, and IL-2 production or [3H]thymidine incorporation by CD4+CD25– cells was measured in the presence of anti-CD3 mAb and increasing numbers of CD4+CD25+ T cells. As shown in Fig. 1, 50% inhibition of the maximum level of IL-2 secreted by both B6 and IBN-Tg conventional cells occurred at a ratio of 0.25 regulatory cells to 1 responding cell regardless of the type (wild-type or IBN-Tg) of CD4+CD25+ or CD4+CD25– cell used. Similar results were observed when proliferation was assayed in anti-CD3-stimulated cultures (Fig. 2) as well as in cultures stimulated with irradiated allogeneic BALB/c splenocytes (data not shown). These results indicate that IBN-Tg Tregs have similar suppressor ability as wild-type CD4+CD25+ cells, and that IBN-Tg CD4+CD25– T cells have similar susceptibility to suppression as wild-type cells. Finally, similar results were found when T cells from tolerant, rather than unmanipulated, IBN-Tg mice were used as the source of CD4+CD25+ and CD4+CD25– T cells (Figs. 3 and 4), suggesting that the state of tolerance does not correlate with a global increase in the total number or function of CD4+CD25+ IBN-Tg T cells. Taken together, these results indicate that reduced NF-B activation in T cells does not result in increased intrinsic or induced suppressor function by CD4+CD25+ cells or increased susceptibility to suppression of CD4+CD25– T cells.
FIGURE 1. T cells from naive IBN-Tg mice do not have increased capacity to suppress IL-2 or augmented susceptibility to suppression. CD4+CD25– and CD4+CD25– cells were sorted from the spleen of B6 or unmanipulated IBN-Tg mice. CD4+CD25– T cells were stimulated with soluble anti-CD3 and irradiated syngeneic B6 splenocytes in the presence of increasing numbers of CD4+CD25+ cells. Supernatants were collected at 24 h and analyzed for IL-2 content by ELISA. The plot represents the mean and SD of triplicate determinations. This result is representative of three independent experiments.
FIGURE 2. T cells from naive IBN-Tg mice do not have increased capacity to suppress proliferation. The experiment was performed as described in Fig. 1, but plates were incubated for 72 h and pulsed with [3H]thymidine for the last 8 h of culture.
FIGURE 3. T cells from tolerant IBN-Tg mice do not have increased capacity to suppress IL-2 or augmented susceptibility to suppression. CD4+CD25– and CD4+CD25– cells were sorted from the spleen of unmanipulated B6 or of IBN-Tg mice that had received a BALB/c heart >50 days previously. Cells were assayed as described in Fig. 1.
FIGURE 4. T cells from tolerant IBN-Tg mice do not have increased capacity to suppress proliferation. The experiment was performed as described in Fig. 3, but plates were incubated for 72 h and pulsed with [3H]thymidine for the last 8 h of the culture.
Tolerance in IBN-Tg mice cannot be transferred to fresh wild-type T cells
Although the proportion and function of CD4+CD25+ T cells were normal in IBN-Tg mice, it was possible that the tolerance observed in these mice was due to a different subset of spontaneously arising or induced regulatory cells. Thus, to assess whether these transplanted mice had developed regulatory cells capable of suppressing the function of conventional T cells, we investigated whether splenocytes from tolerant mice could suppress the rejection mediated by fresh wild-type splenocytes in an adoptive transfer model. As shown in Fig. 5, left panel, splenocytes from wild-type, but not from tolerant, IBN-Tg mice were capable of inducing rejection of BALB/c hearts when transferred into syngeneic RAG1-KO recipients. Notably, the rejection capacity of wild-type splenocytes was not suppressed by addition of splenocytes from tolerant mice at a 1:1 ratio, suggesting the lack of strong regulation in the spleens of tolerant IBN-Tg mice. As a positive control for these experiments, we used B6 mice transplanted with BALB/c hearts and immunosuppressed with a combination of anti-CD40L mAb and perioperative injection of donor splenocytes (anti-CD40L+DST). This treatment resulted in long-term cardiac allograft acceptance (data not shown). In contrast to the lack of suppression of fresh B6 splenocytes by splenocytes from tolerant IBN-Tg mice, splenocytes from anti-CD40L+DST mice effectively reduced the capacity of B6 splenocytes to reject a donor heart in RAG1-KO recipient mice (Fig. 5, right panel).
FIGURE 5. Fresh, wild-type splenocytes are not suppressed by tolerant IBN-Tg splenocytes in RAG1-KO recipients. Splenocytes from unmanipulated B6 (n = 4, left panel; n = 3, right panel), from IBN-Tg mice that had accepted a BALB/c heart for >50 days (left panel; alone, n = 3; with B6 splenocytes at a 1:1 ratio, n = 6), or from B6 mice treated with anti-CD40L and DST that had accepted a BALB/c heart for >43 days (right panel; alone, n = 3; with B6 splenocytes at a 1:1 ratio, n = 7) were adoptively transferred into syngeneic RAG1-deficient mice 1 day after the transplantation of a BALB/c heart. Graft survival in the transferred mice was assessed over time.
It remained possible that the regulation, if any, was not contained in the spleen of tolerant mice. Therefore, to address whether tolerant IBN-Tg mice could suppress the function of wild-type cells, a different adoptive transfer model was designed. Unmanipulated IBN-Tg mice or IBN-Tg mice that had been transplanted with BALB/c hearts >50 days previously were transferred with wild-type splenocytes at the time of transplantation of a second fresh BALB/c heart. The second heart was transplanted to avoid concerns that putative lack of rejection of the original heart may be due to graft adaptation over time. A dose of 80 x 106 splenocytes was chosen, because this was the minimum number of cells that consistently promoted cardiac allograft rejection when transferred into naive, freshly transplanted IBN-Tg mice. As shown in Table II, wild-type splenocytes effectively promoted rejection of fresh BALB/c hearts whether transferred into naive or tolerant IBN-Tg mice, indicating that regulation, if it exists in these mice, is not strong enough to suppress wild-type splenocytes. In contrast to the fresh second set of heart transplants that was rejected, some first-set heart grafts were retained after the adoptive transfer of fresh splenocytes, suggesting that attrition of passenger leukocytes or graft adaptation may have indeed occurred in those long-term accepted transplants. In contrast, B6 mice having accepted an initial BALB/c heart after anti-CD40L+DST treatment retained a second fresh donor heart transplant after transfer of fresh B6 splenocytes, indicating that in some models of long-term graft acceptance, dominant suppression does develop.
Table II. Fresh wild-type splenocytes are not suppressed in tolerant IBN-Tg micea
One hallmark of dominant tolerance is that mice tolerant to Ag A will accept skin grafts from AxB mice, which coexpress the initial Ag to which tolerance was induced and a new Ag to which the recipient mice are naive (20). To test whether tolerant IBN-Tg mice can develop this linked suppression, IBN-Tg mice that had accepted a BALB/c heart for >50 days were transplanted with BALB/c x A/J F1 skin grafts. All transplanted animals rapidly rejected these skin grafts, whereas they retained their BALB/c cardiac allografts (data not shown). Taken together with the adoptive transfer experiments, these results suggest that dominant suppression is not the major mechanism of tolerance in IBN-Tg mice.
Bcl-xL/IBN-Tg mice reject cardiac allografts
Aside from regulation, another major mechanism of allograft tolerance that has been uncovered in certain transplantation models is that of deletion of allospecific T cells (21). Because NF-B activation results in the transcription of several genes involved in cell survival, including A1, A20, inhibitor of apoptosis, and Bcl-xL, it was possible that NF-B-deficient mice would achieve transplantation tolerance because allospecific T cells would die upon antigenic stimulation secondary to the absence of prosurvival proteins up-regulation. Overexpression of Bcl-xL in T cells has been shown to protect T cells from apoptosis induced by TCR stimulation (22). Therefore, we crossed Bcl-xL-Tg (H-2b) with IBN-Tg (H-2b) mice and transplanted the resulting single- and double-transgenic animals with BALB/c hearts. T cells from double-transgenic mice have also been shown to have reduced susceptibility to apoptosis (23). As shown in Fig. 6A, although the majority of IBN-Tg littermates accepted BALB/c hearts long-term, all Bcl-xL/IBN-Tg mice rejected cardiac allografts, albeit with delayed kinetics compared with Bcl-xL-Tg control littermates.
FIGURE 6. Expression of a Bcl-xL transgene in T cells prevents tolerance induction in IBN-Tg mice. BALB/c hearts were transplanted into littermates of the different strains. A, Graft survival was assessed over time (wild-type, n = 7; Bcl-xL-Tg, n = 3; IBN-Tg, n = 8; IBN/Bcl-xL-Tg littermates, n = 9). B, Splenocytes were stimulated with irradiated B6 or BALB/c splenocytes on day 25 posttransplant, and the precursor frequency of IFN--producing cells was determined using an ELISPOT assay. The plot represents the means and SDs of at least triplicate determinations. ***, p < 0.001, as determined by Student’s t test.
To address whether expression of the Bcl-xL transgene promoted survival of allospecific T cells, an IFN--specific ELISPOT assay was performed using splenocytes from mice transplanted with a BALB/c heart 25 days previously. IBN-Tg mice that had accepted BALB/c hearts had few IFN--producing cells. In addition to promoting rejection, expression of the Bcl-xL transgene in IBN-Tg mice resulted in markedly enhanced numbers of IFN--producing cells, almost as high as in NF-B-sufficient mice that had also rejected their grafts (Fig. 6B). This was not due to an increased number of T cells in wells assaying the responsiveness of Bcl-x/IBN-Tg splenocytes, because the percentage of T cells was similar in all IBN-Tg and Bcl-x/IBN-Tg spleens, as determined by flow cytometry (data not shown). This result implies that BALB/c-specific cells are alive and functional in Bcl-x/IBN-Tg mice. Together, these data indicate that overexpression of Bcl-xL in T cells results in rejection of cardiac allografts by mice with impaired T cell-intrinsic NF-B activation and suggest that T cell deletion plays an important role in the tolerance observed in IBN-Tg mice.
Discussion
We have previously reported that mice with reduced T cell-intrinsic NF-B activation permanently accept primary fully allogeneic heart transplants and secondary donor, but not third-party, skin grafts (15), indicating robust donor-specific tolerance. These results identified NF-B in T cells as a possible target for immune modulation in the clinic, but also called for vigorous investigation of the mechanisms by which this tolerance was achieved to evaluate possible clinical applicability. In this study we demonstrate that T cell regulation is not the major mechanism leading to tolerance in this model. Rather, transgenic expression of Bcl-xL in T cells restores cardiac allograft rejection in T cell-intrinsic, NF-B-impaired mice.
Several immunosuppressive regimens have been associated with long-term cardiac allograft acceptance in mice. These include treatment with Abs or fusion proteins thought to block engagement of costimulatory receptors such as CD28/CD80/CD86 (24) or CD40/CD154 (25), administration of nondepleting anti-CD4 and anti-CD8 Abs (26), treatment with gallium nitrate (27), or combination therapies that include donor-specific transfusions (28, 29). The mechanisms by which these treatments induce graft acceptance can be divided into two broad nonexclusive categories: T cell depletion and regulation of T cell responses. For instance, treatment with CTLA-4-Ig or anti-CD154 mAb has been reported to promote T cell depletion (22, 30), but administration of anti-CD154 mAb has also been associated with the development of regulation in a skin graft model (31, 32). Nondepleting anti-CD4 and anti-CD8 mAbs have been shown to promote dominant suppressive tolerance in skin graft models (33), whereas transfusion of donor cells has been reported to promote either regulation (34) or deletion (29) of alloreactive T cells in cardiac or skin transplant models.
Reduced NF-B activation in T cells also results in long-term cardiac allograft acceptance (14, 15). However, we were unable to uncover the existence of a regulatory mechanism in IBN-Tg mice. It was clear that IBN-Tg mice did not have an increased percentage or number of native CD4+CD25+ T cells. In fact, NF-B activation has recently been reported to be necessary for the thymic development of regulatory cells, because mice with either conditional deletion of IKK subunits in T cells or p50/cRel double-deficient mice fail to develop CD4+CD25+ T cells (35, 36). It would appear that the residual level of NF-B activation in T cells from IBN-Tg mice is sufficient for the development of innate regulatory T cells in these mice. Although rejection of cardiac allografts is mainly dependent on the presence of CD4+ and not CD8+ T cells (37, 38, 39, 40, 41), immunohistochemistry of graft tissue at the time of rejection usually reveals a majority of CD8+ T cells, suggesting that this subset is an active, albeit dispensable, participant in the effector phase of the rejection process (42). CD4+CD25+ T cells can suppress the function of CD8+ T cells (43, 44, 45). IBN-Tg mice have markedly reduced numbers of CD8+ T cells (13). Thus, although the proportion of CD4+CD25+ T cells relative to the number of CD4+ T cells appears normal in IBN-Tg mice, the ratio of Treg to CD8+ T cells is indeed increased. Therefore, although the suppressor capacity of IBN-Tg CD4+CD25+ cells is not increased on a per cell basis, we cannot exclude the possibility that these mice have a greater capacity to regulate their CD8 effector T cell responses in vivo.
Several groups have now shown that CD4+CD25+ T cells that suppress allogeneic responses in vivo can arise from CD4+CD25– T cells after transplantation (46, 47). However, we did not find increased numbers of CD4+CD25+ T cells in tolerant IBN-Tg mice (data not shown). Aside from CD4+CD25+ cells, other subsets of T cells, such as NKT cells, some subpopulations of CD8+ T cells, and induced T regulatory 1 or Th3 cells, can also suppress T cell responses in some settings (48). Thus, it was possible that reduced NF-B activation in T cells would affect the development of other regulatory T cell subsets in IBN-Tg mice or prompt the development of induced regulatory T cells upon encounter of T cells with allogeneic Ags in vivo. Although it was unlikely that regulation would be supported by NKT cells in this model, because NF-B activation in T cells is necessary for the thymic development of this cell type (49, 50), other cell subsets could have been involved. However, we could not find evidence for dominant regulation in tolerant IBN-Tg mice. The lack of strong regulation was concluded because of the concordant results of three lines of experiments: fresh wild-type splenocytes were not suppressed by splenocytes from tolerant IBN-Tg mice in RAG1-deficient mice; fresh wild-type splenocytes were not suppressed in tolerant IBN-Tg mice transplanted with fresh second donor hearts; tolerant IBN-Tg mice accepted donor skin, but rejected F1 skin grafts that expressed donor Ags. We cannot exclude that tolerant IBN-Tg mice have developed a low level of regulation that we are unable to detect with our assays. For instance, although a 1:1 ratio of tolerant to wild-type fresh splenocytes did not prevent wild-type splenocyte-mediated cardiac allograft rejection in RAG1-KO mice, it is possible that a greater ratio would be successful. Nevertheless, we conclude that if regulation exists in this model, it is not dominant, not easily transferable, and of lower magnitude than that induced in B6 mice by anti-CD40L + DST treatment.
In contrast with the lack of evidence for dominant suppression, it was clear that Bcl-xL/IBN-Tg mice were capable of rejecting cardiac allografts. Although NF-B promotes the transcription of multiple antiapoptotic genes, including Bcl-xL, overexpression of just Bcl-xL has been shown to be sufficient to reduce the death of IBN-Tg T cells (23). However, it is also possible that transgenic expression of Bcl-xL in T cells modifies aspects of T cell biology other than survival, and that cardiac allograft rejection in Bcl-xL/IBN-Tg mice is not due to prevention of death of IBN-Tg T cells, but, rather, to increased T cell effector function, for instance. Similar to our model, overexpression of Bcl-xL in T cells has previously been reported to prevent tolerance induction in mice treated with a combination of donor-specific transfusion with CTLA-4-Ig or anti-CD154 (22). These results support the idea that reducing T cell death can prevent transplantation tolerance in models in which apoptosis is the major mechanism of tolerance, although in neither case can it be excluded that Bcl-xL is operating through alternative mechanisms than prevention of apoptosis. Future experiments using TCR transgenic/IBN-Tg mice to be able to follow the fate of alloreactive T cells after transplantation will help determine whether the effect of Bcl-xL depends on prevention of T cell death.
The importance of the precursor frequency of graft-specific T cells in the success or failure to reject a transplant is becoming increasingly apparent (51, 52). Because regulation of alloresponses can coexist with a certain level of deletion of alloreactive T cells, it has been suggested that it may be possible to tip the balance of these two mechanisms in favor of transplantation tolerance (53). In fact, in the absence of regulation or constant depletion of new emerging T cells, T cell deletion may impede transplantation tolerance as new developing T cells may undergo homeostatic expansion to reconstitute the T cell repertoire and thus acquire memory characteristics that confer resistance to certain regimens of tolerance induction, such as costimulatory blockade (54). It is possible that IBN-Tg mice remain tolerant indefinitely without histological evidence of chronic graft rejection (14) because of the continuous deletion of new emerging alloreactive T cells as they encounter alloantigen in the presence of blunted NF-B activation.
The pathway that leads to NF-B activation in T cells downstream of TCR/CD28 engagement is unique. It involves recruitment and activation of protein kinase C, the adaptors CARMA1, Bcl10, and MALT1 and the recruitment of a ubiquitinating complex that includes TRAF6 and results in activation of TAK1, followed by activation of the IKK complex (55). Protein kinase C is mostly restricted to T cells, whereas CARMA1, Bcl10, and MALT1 appear lymphocyte-restricted. Therefore, it may be possible to develop small molecule inhibitors that are cell-permeable and inhibit the binding or activation of these lymphocyte-restricted proteins. These therapies should only inhibit TCR- or BCR-mediated NF-B activation and may reproduce in normal mice or patients the susceptibility to tolerance observed in IBN-Tg mice. However, whether transient deletion of alloreactive T cells will be sufficient to induce and maintain tolerance and whether these therapies will be effective to induce deletion of pre-existing cross-reactive memory cells remain to be established.
Disclosures
The authors have no financial conflict of interest.
Acknowledgments
We thank James Marvin and Ryan Duggan for expert help with cell sorting.
Footnotes
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 This work was supported by American Heart Association Grant 0350261N, Juvenile Diabetes Research Foundation Grant 1-2003-188, and National Institutes of Health Grant RO1AI052352-01.
2 P.Z. and S.J.B. contributed equally to this work.
3 Current address: Department of Immunology, College of Pharmacy, Chuang Ang University, Seoul 156756, Korea.
4 Address correspondence and reprint requests to Dr. Maria-Luisa Alegre, University of Chicago, 5841 S. Maryland Avenue, Room N005C, MC 0930, Chicago, IL 60637. E-mail address: malegre{at}midway.uchicago.edu
5 Abbreviations used in this paper: RHD, Rel homology domain; DST, donor-specific transfusion; IKK, IB kinase; RAG1-KO, RAG1-deficient; Treg, regulatory T cell.
Received for publication June 7, 2004. Accepted for publication January 9, 2005.
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NF-B is a key regulator of transcription after TCR and costimulatory receptor ligation. To determine the role of T cell-intrinsic NF-B activation in acute allograft rejection, we used IBN-Tg mice (H-2b) that express an inhibitor of NF-B restricted to the T cell compartment. We have previously shown that these mice permanently accept fully allogeneic (H-2d) cardiac grafts and secondary donor skin grafts, and that splenocytes from these tolerant mice have reduced alloreactivity when restimulated in vitro. These results were compatible with either deletion or suppression of allospecific T cells as possible mechanisms of tolerance. The aim of this study was to investigate the mechanism of transplant tolerance in these mice. IBN-Tg mice did not have increased numbers or function of CD4+CD25+ regulatory T cells either before or after cardiac transplantation. In addition, tolerance could not be transferred to fresh NF-B-competent T cells and was not permissive for linked suppression to skin grafts sharing donor and third-party alloantigens, suggesting that dominant suppression is not the mechanism by which IBN-Tg mice achieve tolerance. In contrast, overexpression of the antiapoptotic protein Bcl-xL in T cells from IBN-Tg mice resulted in effective rejection of cardiac allografts and correlated with an increased frequency of splenocytes producing IFN- in response to alloantigen. Together, these results suggest that the death of alloreactive T cells may be partly responsible for the transplantation tolerance observed in mice with defective T cell-intrinsic NF-B activation.
Introduction
Solid organ transplantation is often the only cure for end-stage organ failure. However, unless tissues are donated by identical twins, transplantation is limited by the occurrence of acute allograft rejection. Acute allograft rejection is mediated by T lymphocytes, and its prevention requires transplant recipients to take life-long immunosuppressive therapies, with the potential complications of infections and virus-induced tumors that are common with chronic immunosuppression. Therefore, the goal of transplantation research is to develop immunosuppressive regimens that inactivate only alloreactive T cells, leaving the rest of the immune system competent to react against pathogens and tumor Ags, a state termed donor-specific tolerance. Thus, understanding the biochemical pathways in T cells that are necessary to mount acute allograft rejection episodes is essential for the design of such novel therapies.
T cell activation follows engagement by the TCR of specific peptide/MHC complexes displayed on the surface of APCs, concurrently to the ligation of coreceptors (CD4 or CD8) and costimulatory receptors on T cells. An essential costimulatory receptor for naive T cells is CD28, which binds B7-1 (CD80) and B7-2 (CD86) on APCs (1). Engagement of TCR in the absence of CD28 ligation results in T cell death or T cell inactivation (2, 3). Biochemical signals transduced upon TCR/CD28 engagement include activation of the Src kinase Lck, phosphorylation of ITAM motifs in TCR -chains, and recruitment and activation of the tyrosine kinase ZAP70 and subsequently of the scaffolding adaptors Lat and SLP76 that are essential for the formation of signalosomes localized to lipid rafts at the plasma membrane (4). These signalosomes promote activation of downstream transcription factors that in naive T cells include NFAT, AP-1, and NF of the B cell -chain (NF-B). Although these events have been well established in vitro, it is not clear whether all these pathways take place after T cell activation in vivo or what steps are necessary for mounting a productive immune response in the context of transplant rejection. Several groups have begun investigating the role of transcription factors during acute allograft rejection. For instance, inhibition of NFAT using cell-permeable inhibitory peptides resulted in prolongation of islet allograft survival in mice (5).
Our laboratory has chosen to concentrate on the role of NF-B activation in T cells during acute allograft rejection, because NF-B had been implicated in cell survival, proliferation, and cytokine production in several model systems in vitro (6) and therefore was likely to play an important role in immune responses in vivo. The Rel/NF-B family of proteins is composed of five members that each contain a Rel homology domain (RHD)5 important for protein and DNA binding. p50 and p52 bind DNA well, but have weak transactivating capacity, whereas RelA (p65), RelB, and c-Rel are poor DNA binders, but contain a DNA transactivating domain. Typical NF-B molecules are composed of a heterodimeric combination of p50 or p52, bound with p65, RelB, or c-Rel. T cells contain all NF-B family subunits. In resting cells, NF-B dimers are sequestered in the cytoplasm by IB family members. IB molecules bind to and cover the nuclear localization signals on NF-B heterodimers, thus preventing their translocation to the nucleus (6). Engagement of TCR and CD28 results in activation of IB kinases (IKK), which phosphorylate IB. The phosphorylated IB is then ubiquitinated and subsequently degraded by the proteasome. This exposes the nuclear localization signal on the NF-B dimer, which can move into the nucleus and activate gene transcription. Nuclear translocation of NF-B subunits has been reported in cardiac allografts at the time of T cell infiltration and initiation of rejection (7).
Many immunosuppressive drugs commonly used to prevent or treat allograft rejection inhibit NF-B as part of their mechanism of action, although they may also exert many other effects. Corticosteroids, for instance, induce de novo transcription of IB molecules and therefore inhibit further NF-B activation (8, 9). Several nonsteroidal anti-inflammatory drugs reduce the phosphorylation of IB by the IKK complex (10). Even cyclosporin A, a compound known for its inhibitory activity of NFAT cells, has been shown to reduce proteasomal degradation of phosphorylated IBs (10). The immunosuppressive activity of these drugs is probably due at least in part to their NF-B inhibitory effects.
Transplantation experiments have been performed on mice genetically deficient in single NF-B subunits, but prolongation of graft survival in these mice has been slight, presumably due to redundancy and compensation by other subunits. Thus, p50- or p52-deficient mice effectively reject cardiac allografts (11, 12). The role of T cell-intrinsic NF-B activation during allograft rejection has been reinvestigated using mice that have reduced NF-B activity selectively in T cells. These mice express a super-repressor transgenic form of IB driven by the Lck proximal promoter and CD2 locus control region (13). This transgene bears a deletion of the region containing the serines that serve as targets for the IKK complex. Thus, this mutant IB cannot be phosphorylated by the IKK complex and therefore retains the NF-B subunits to which it is bound in the cytoplasm. We and others have shown that the majority of IBN-Tg mice permanently accept heart transplants (14, 15), but reject skin allografts (15). However, IBN-Tg mice tolerant to cardiac allografts permanently accept secondary donor, but not third-party, skin allografts, indicative of donor-specific tolerance (15). Splenocytes from these mice had reduced reactivity against donor, but not third-party, Ags (15), suggesting that alloreactive T cells had been deleted or were hyporesponsive either because they had become anergic or because they were being suppressed by regulatory cells. Several transplantation models in which long-term graft survival has been achieved have identified either T cell apoptosis or T cell regulation as a major mechanism to attenuate rejection (16). In this study we have investigated whether graft acceptance in IBN-Tg mice is due to regulation or deletion of alloreactive T cells. Tolerance in this model was not transferable to fresh wild-type T cells. In contrast, overexpression in T cells of the antiapoptotic protein Bcl-xL resulted in cardiac allograft rejection by IBN-Tg mice, suggesting that apoptosis, but not regulation, may be the main mechanistic pathway of tolerance in these mice.
Materials and Methods
Mice
IBN-Tg mice (H-2b) have been previously described (13) and were a gift from M. Boothby (Vanderbilt University, Nashville, TE). Bcl-xL-Tg mice (H-2b) have been previously described (17) and were provided by C. Thompson (University of Pennsylvania, Philadelphia, PA). IBN-Tg and Bcl-xL-Tg mice were intercrossed to generate double-transgenic mice (Bcl-xL/IBN-Tg). C57BL/6 (B6; H-2b) and BALB/c (H-2d) were purchased from Frederick Cancer Research Facilities. B6/RAG1-deficient (RAG1-KO; H-2b) mice were bred in our animal facility. Animals were housed in ventilated racks in a specific pathogen-free animal facility. Experiments were performed in agreement with our Institutional animal care and use committee and according to the National Institutes of Health guidelines for animal use.
Heart and skin transplantation
Abdominal heterotopic cardiac transplantation was performed using a technique adapted from that originally described by Corry et al. (18). Cardiac allografts were transplanted in the abdominal cavity by anastomosing the aorta and pulmonary artery of the graft end-to-side to the recipient’s aorta and vena cava, respectively. The day of rejection was defined as the last day of a detectable heartbeat in the graft. Graft rejection was verified in selected cases by necropsy and pathological examination of H&E-stained graft sections.
Cervical cardiac transplantation was performed using a technique adapted from Chen (19). The cardiac graft was placed under the skin of the front of the neck. The innominate artery of the donor heart was anastomosed end-to-end to the right common carotid artery of the recipient. The pulmonary artery of the donor heart was anastomosed end-to-end to the external jugular vein of the recipient.
Some B6 mice were transplanted with BALB/c hearts and treated with a combination of anti-CD40L (MR1, 1 mg i.p. on days 0, 7, and 14 posttransplantation; Ligocyte Pharmaceuticals) and donor-specific transfusion (DST; 5 x 106 BALB/c splenocytes i.v. on day 0).
Flow cytometry
Splenocytes were isolated and processed into single cell suspensions. Cells were stained with anti-CD4-FITC- and anti-CD25-PE-coupled Abs (BD Pharmingen) and analyzed by flow cytometry (FACScan I or II; BD Biosciences).
Functional suppressor capacity of regulatory T cells (Tregs)
To assess the regulatory capacity of CD4+CD25+ T cells, splenocytes from unmanipulated B6 and IBN-Tg mice either unmanipulated or transplanted >50 days previously with a BALB/c heart were isolated and stained with dialyzed anti-CD4-FITC and anti-CD25-PE Abs. Cell populations were sorted using a MOFLO-HTS cell sorter (DakoCytomation) into CD4+CD25– (responding cells) or CD4+CD25+ (regulatory cells). Responding cells were resuspended in DMEM supplemented with 10% FBS, penicillin (100 U/ml), streptomycin (100 μg/ml), HEPES, 2-ME (50 μM), and additional amino acids and seeded in 96-well, round-bottom plates (3 x 104 cells/well) in the presence of soluble anti-CD3 (10 μg/ml) and irradiated (2000 rad) syngeneic splenocytes (15 x 104 cells/well). Increasing numbers of CD4+CD25+ T cells were added to the wells. Supernatants were collected at 24 h, and the concentration of IL-2 was measured by ELISA using Ab pairs (BD Pharmingen). Parallel plates were pulsed with [3H]thymidine for the last 8 h of a 72-h culture (1 μCi/well).
Adoptive transfer of T cells
Spleens were harvested from unmanipulated B6 mice or anti-CD40L- plus DST-treated, long-term BALB/c cardiac graft-accepting B6 mice or IBN-Tg mice that had accepted a BALB/c heart transplanted >50 days previously. Indicated numbers of splenocytes were resuspended in 150 μl of PBS and injected i.v. into the retro-orbital plexus of recipient mice. Recipient mice in some experiments were syngeneic RAG1-KO mice that had been transplanted 1 day previously with BALB/c cardiac allografts. In other experiments, recipient mice were anti-CD40L- plus DST-treated, long-term BALB/c cardiac graft-accepting B6 mice or IBN-Tg mice that had accepted a cervical BALB/c heart for >50 days. These recipients received a second abdominal BALB/c cardiac transplant 1 day before the adoptive transfer.
IFN- ELISPOTs
Splenocytes from untransplanted mice or from mice transplanted 25 days previously with a BALB/c heart (106/well) were restimulated in vitro with irradiated B6 or BALB/c splenocytes (4 x 105/well) and incubated for 24 h in a 5% CO2 incubator. The ELISPOT assay was conducted according to the instructions of the manufacturer (BD Biosciences), and the numbers of spots per well were calculated using the ImmunoSpot Analyzer (CTL Analyzers LLC).
Results
IBN-Tg mice do not have increased numbers of CD4+CD25+ regulatory T cells either before or after heart allograft acceptance
CD4+CD25+ T cells are naturally occurring regulatory cells that develop in the thymus. It was possible that reduced NF-B activation during thymic maturation promoted the development of Treg at the expense of effector T cells, so that the ratio of CD4+CD25+ to CD4+CD25– T cells be altered and favor tolerance induction. To test this hypothesis, we compared the number of CD4+CD25+ T cells in wild-type and IBN-Tg littermates. Consistent with the known reduction in the number of total CD4+ T cells in IBN-Tg mice compared with wild-type mice, the total number of CD4+CD25+ was also reduced in the spleens of these mice (Table I). However, the percentage of CD4+CD25+ within the CD4+ population was similar to that in wild-type mice in the spleen of both unmanipulated (Table I) and tolerant (data not shown) IBN-Tg mice. Similar results were obtained in lymph nodes of these animals (data not shown). This result indicates that reduced NF-B activation in T cells does not favor the development of a greater proportion of CD4+CD25+ T cells either spontaneously or after alloantigenic encounter.
Table I. IBN-Tg mice do not have an increased number of CD4+CD25+ cellsa
T cells from both unmanipulated and tolerant IBN-Tg mice do not have an intrinsically better capacity to regulate or be regulated than wild-type T cells
Although the number of CD4+CD25+ T cells was not increased in IBN-Tg mice when compared with wild-type mice, it was possible that IBN-Tg CD4+CD25+ T cells had increased suppressor function on a per cell basis or that IBN-Tg CD4+CD25– cells were more susceptible to suppression than wild-type T cells. To explore these possibilities, CD4+ cells from unmanipulated IBN-Tg and control littermates mice were sorted into populations of CD4+CD25+ and CD4+CD25– cells, and IL-2 production or [3H]thymidine incorporation by CD4+CD25– cells was measured in the presence of anti-CD3 mAb and increasing numbers of CD4+CD25+ T cells. As shown in Fig. 1, 50% inhibition of the maximum level of IL-2 secreted by both B6 and IBN-Tg conventional cells occurred at a ratio of 0.25 regulatory cells to 1 responding cell regardless of the type (wild-type or IBN-Tg) of CD4+CD25+ or CD4+CD25– cell used. Similar results were observed when proliferation was assayed in anti-CD3-stimulated cultures (Fig. 2) as well as in cultures stimulated with irradiated allogeneic BALB/c splenocytes (data not shown). These results indicate that IBN-Tg Tregs have similar suppressor ability as wild-type CD4+CD25+ cells, and that IBN-Tg CD4+CD25– T cells have similar susceptibility to suppression as wild-type cells. Finally, similar results were found when T cells from tolerant, rather than unmanipulated, IBN-Tg mice were used as the source of CD4+CD25+ and CD4+CD25– T cells (Figs. 3 and 4), suggesting that the state of tolerance does not correlate with a global increase in the total number or function of CD4+CD25+ IBN-Tg T cells. Taken together, these results indicate that reduced NF-B activation in T cells does not result in increased intrinsic or induced suppressor function by CD4+CD25+ cells or increased susceptibility to suppression of CD4+CD25– T cells.
FIGURE 1. T cells from naive IBN-Tg mice do not have increased capacity to suppress IL-2 or augmented susceptibility to suppression. CD4+CD25– and CD4+CD25– cells were sorted from the spleen of B6 or unmanipulated IBN-Tg mice. CD4+CD25– T cells were stimulated with soluble anti-CD3 and irradiated syngeneic B6 splenocytes in the presence of increasing numbers of CD4+CD25+ cells. Supernatants were collected at 24 h and analyzed for IL-2 content by ELISA. The plot represents the mean and SD of triplicate determinations. This result is representative of three independent experiments.
FIGURE 2. T cells from naive IBN-Tg mice do not have increased capacity to suppress proliferation. The experiment was performed as described in Fig. 1, but plates were incubated for 72 h and pulsed with [3H]thymidine for the last 8 h of culture.
FIGURE 3. T cells from tolerant IBN-Tg mice do not have increased capacity to suppress IL-2 or augmented susceptibility to suppression. CD4+CD25– and CD4+CD25– cells were sorted from the spleen of unmanipulated B6 or of IBN-Tg mice that had received a BALB/c heart >50 days previously. Cells were assayed as described in Fig. 1.
FIGURE 4. T cells from tolerant IBN-Tg mice do not have increased capacity to suppress proliferation. The experiment was performed as described in Fig. 3, but plates were incubated for 72 h and pulsed with [3H]thymidine for the last 8 h of the culture.
Tolerance in IBN-Tg mice cannot be transferred to fresh wild-type T cells
Although the proportion and function of CD4+CD25+ T cells were normal in IBN-Tg mice, it was possible that the tolerance observed in these mice was due to a different subset of spontaneously arising or induced regulatory cells. Thus, to assess whether these transplanted mice had developed regulatory cells capable of suppressing the function of conventional T cells, we investigated whether splenocytes from tolerant mice could suppress the rejection mediated by fresh wild-type splenocytes in an adoptive transfer model. As shown in Fig. 5, left panel, splenocytes from wild-type, but not from tolerant, IBN-Tg mice were capable of inducing rejection of BALB/c hearts when transferred into syngeneic RAG1-KO recipients. Notably, the rejection capacity of wild-type splenocytes was not suppressed by addition of splenocytes from tolerant mice at a 1:1 ratio, suggesting the lack of strong regulation in the spleens of tolerant IBN-Tg mice. As a positive control for these experiments, we used B6 mice transplanted with BALB/c hearts and immunosuppressed with a combination of anti-CD40L mAb and perioperative injection of donor splenocytes (anti-CD40L+DST). This treatment resulted in long-term cardiac allograft acceptance (data not shown). In contrast to the lack of suppression of fresh B6 splenocytes by splenocytes from tolerant IBN-Tg mice, splenocytes from anti-CD40L+DST mice effectively reduced the capacity of B6 splenocytes to reject a donor heart in RAG1-KO recipient mice (Fig. 5, right panel).
FIGURE 5. Fresh, wild-type splenocytes are not suppressed by tolerant IBN-Tg splenocytes in RAG1-KO recipients. Splenocytes from unmanipulated B6 (n = 4, left panel; n = 3, right panel), from IBN-Tg mice that had accepted a BALB/c heart for >50 days (left panel; alone, n = 3; with B6 splenocytes at a 1:1 ratio, n = 6), or from B6 mice treated with anti-CD40L and DST that had accepted a BALB/c heart for >43 days (right panel; alone, n = 3; with B6 splenocytes at a 1:1 ratio, n = 7) were adoptively transferred into syngeneic RAG1-deficient mice 1 day after the transplantation of a BALB/c heart. Graft survival in the transferred mice was assessed over time.
It remained possible that the regulation, if any, was not contained in the spleen of tolerant mice. Therefore, to address whether tolerant IBN-Tg mice could suppress the function of wild-type cells, a different adoptive transfer model was designed. Unmanipulated IBN-Tg mice or IBN-Tg mice that had been transplanted with BALB/c hearts >50 days previously were transferred with wild-type splenocytes at the time of transplantation of a second fresh BALB/c heart. The second heart was transplanted to avoid concerns that putative lack of rejection of the original heart may be due to graft adaptation over time. A dose of 80 x 106 splenocytes was chosen, because this was the minimum number of cells that consistently promoted cardiac allograft rejection when transferred into naive, freshly transplanted IBN-Tg mice. As shown in Table II, wild-type splenocytes effectively promoted rejection of fresh BALB/c hearts whether transferred into naive or tolerant IBN-Tg mice, indicating that regulation, if it exists in these mice, is not strong enough to suppress wild-type splenocytes. In contrast to the fresh second set of heart transplants that was rejected, some first-set heart grafts were retained after the adoptive transfer of fresh splenocytes, suggesting that attrition of passenger leukocytes or graft adaptation may have indeed occurred in those long-term accepted transplants. In contrast, B6 mice having accepted an initial BALB/c heart after anti-CD40L+DST treatment retained a second fresh donor heart transplant after transfer of fresh B6 splenocytes, indicating that in some models of long-term graft acceptance, dominant suppression does develop.
Table II. Fresh wild-type splenocytes are not suppressed in tolerant IBN-Tg micea
One hallmark of dominant tolerance is that mice tolerant to Ag A will accept skin grafts from AxB mice, which coexpress the initial Ag to which tolerance was induced and a new Ag to which the recipient mice are naive (20). To test whether tolerant IBN-Tg mice can develop this linked suppression, IBN-Tg mice that had accepted a BALB/c heart for >50 days were transplanted with BALB/c x A/J F1 skin grafts. All transplanted animals rapidly rejected these skin grafts, whereas they retained their BALB/c cardiac allografts (data not shown). Taken together with the adoptive transfer experiments, these results suggest that dominant suppression is not the major mechanism of tolerance in IBN-Tg mice.
Bcl-xL/IBN-Tg mice reject cardiac allografts
Aside from regulation, another major mechanism of allograft tolerance that has been uncovered in certain transplantation models is that of deletion of allospecific T cells (21). Because NF-B activation results in the transcription of several genes involved in cell survival, including A1, A20, inhibitor of apoptosis, and Bcl-xL, it was possible that NF-B-deficient mice would achieve transplantation tolerance because allospecific T cells would die upon antigenic stimulation secondary to the absence of prosurvival proteins up-regulation. Overexpression of Bcl-xL in T cells has been shown to protect T cells from apoptosis induced by TCR stimulation (22). Therefore, we crossed Bcl-xL-Tg (H-2b) with IBN-Tg (H-2b) mice and transplanted the resulting single- and double-transgenic animals with BALB/c hearts. T cells from double-transgenic mice have also been shown to have reduced susceptibility to apoptosis (23). As shown in Fig. 6A, although the majority of IBN-Tg littermates accepted BALB/c hearts long-term, all Bcl-xL/IBN-Tg mice rejected cardiac allografts, albeit with delayed kinetics compared with Bcl-xL-Tg control littermates.
FIGURE 6. Expression of a Bcl-xL transgene in T cells prevents tolerance induction in IBN-Tg mice. BALB/c hearts were transplanted into littermates of the different strains. A, Graft survival was assessed over time (wild-type, n = 7; Bcl-xL-Tg, n = 3; IBN-Tg, n = 8; IBN/Bcl-xL-Tg littermates, n = 9). B, Splenocytes were stimulated with irradiated B6 or BALB/c splenocytes on day 25 posttransplant, and the precursor frequency of IFN--producing cells was determined using an ELISPOT assay. The plot represents the means and SDs of at least triplicate determinations. ***, p < 0.001, as determined by Student’s t test.
To address whether expression of the Bcl-xL transgene promoted survival of allospecific T cells, an IFN--specific ELISPOT assay was performed using splenocytes from mice transplanted with a BALB/c heart 25 days previously. IBN-Tg mice that had accepted BALB/c hearts had few IFN--producing cells. In addition to promoting rejection, expression of the Bcl-xL transgene in IBN-Tg mice resulted in markedly enhanced numbers of IFN--producing cells, almost as high as in NF-B-sufficient mice that had also rejected their grafts (Fig. 6B). This was not due to an increased number of T cells in wells assaying the responsiveness of Bcl-x/IBN-Tg splenocytes, because the percentage of T cells was similar in all IBN-Tg and Bcl-x/IBN-Tg spleens, as determined by flow cytometry (data not shown). This result implies that BALB/c-specific cells are alive and functional in Bcl-x/IBN-Tg mice. Together, these data indicate that overexpression of Bcl-xL in T cells results in rejection of cardiac allografts by mice with impaired T cell-intrinsic NF-B activation and suggest that T cell deletion plays an important role in the tolerance observed in IBN-Tg mice.
Discussion
We have previously reported that mice with reduced T cell-intrinsic NF-B activation permanently accept primary fully allogeneic heart transplants and secondary donor, but not third-party, skin grafts (15), indicating robust donor-specific tolerance. These results identified NF-B in T cells as a possible target for immune modulation in the clinic, but also called for vigorous investigation of the mechanisms by which this tolerance was achieved to evaluate possible clinical applicability. In this study we demonstrate that T cell regulation is not the major mechanism leading to tolerance in this model. Rather, transgenic expression of Bcl-xL in T cells restores cardiac allograft rejection in T cell-intrinsic, NF-B-impaired mice.
Several immunosuppressive regimens have been associated with long-term cardiac allograft acceptance in mice. These include treatment with Abs or fusion proteins thought to block engagement of costimulatory receptors such as CD28/CD80/CD86 (24) or CD40/CD154 (25), administration of nondepleting anti-CD4 and anti-CD8 Abs (26), treatment with gallium nitrate (27), or combination therapies that include donor-specific transfusions (28, 29). The mechanisms by which these treatments induce graft acceptance can be divided into two broad nonexclusive categories: T cell depletion and regulation of T cell responses. For instance, treatment with CTLA-4-Ig or anti-CD154 mAb has been reported to promote T cell depletion (22, 30), but administration of anti-CD154 mAb has also been associated with the development of regulation in a skin graft model (31, 32). Nondepleting anti-CD4 and anti-CD8 mAbs have been shown to promote dominant suppressive tolerance in skin graft models (33), whereas transfusion of donor cells has been reported to promote either regulation (34) or deletion (29) of alloreactive T cells in cardiac or skin transplant models.
Reduced NF-B activation in T cells also results in long-term cardiac allograft acceptance (14, 15). However, we were unable to uncover the existence of a regulatory mechanism in IBN-Tg mice. It was clear that IBN-Tg mice did not have an increased percentage or number of native CD4+CD25+ T cells. In fact, NF-B activation has recently been reported to be necessary for the thymic development of regulatory cells, because mice with either conditional deletion of IKK subunits in T cells or p50/cRel double-deficient mice fail to develop CD4+CD25+ T cells (35, 36). It would appear that the residual level of NF-B activation in T cells from IBN-Tg mice is sufficient for the development of innate regulatory T cells in these mice. Although rejection of cardiac allografts is mainly dependent on the presence of CD4+ and not CD8+ T cells (37, 38, 39, 40, 41), immunohistochemistry of graft tissue at the time of rejection usually reveals a majority of CD8+ T cells, suggesting that this subset is an active, albeit dispensable, participant in the effector phase of the rejection process (42). CD4+CD25+ T cells can suppress the function of CD8+ T cells (43, 44, 45). IBN-Tg mice have markedly reduced numbers of CD8+ T cells (13). Thus, although the proportion of CD4+CD25+ T cells relative to the number of CD4+ T cells appears normal in IBN-Tg mice, the ratio of Treg to CD8+ T cells is indeed increased. Therefore, although the suppressor capacity of IBN-Tg CD4+CD25+ cells is not increased on a per cell basis, we cannot exclude the possibility that these mice have a greater capacity to regulate their CD8 effector T cell responses in vivo.
Several groups have now shown that CD4+CD25+ T cells that suppress allogeneic responses in vivo can arise from CD4+CD25– T cells after transplantation (46, 47). However, we did not find increased numbers of CD4+CD25+ T cells in tolerant IBN-Tg mice (data not shown). Aside from CD4+CD25+ cells, other subsets of T cells, such as NKT cells, some subpopulations of CD8+ T cells, and induced T regulatory 1 or Th3 cells, can also suppress T cell responses in some settings (48). Thus, it was possible that reduced NF-B activation in T cells would affect the development of other regulatory T cell subsets in IBN-Tg mice or prompt the development of induced regulatory T cells upon encounter of T cells with allogeneic Ags in vivo. Although it was unlikely that regulation would be supported by NKT cells in this model, because NF-B activation in T cells is necessary for the thymic development of this cell type (49, 50), other cell subsets could have been involved. However, we could not find evidence for dominant regulation in tolerant IBN-Tg mice. The lack of strong regulation was concluded because of the concordant results of three lines of experiments: fresh wild-type splenocytes were not suppressed by splenocytes from tolerant IBN-Tg mice in RAG1-deficient mice; fresh wild-type splenocytes were not suppressed in tolerant IBN-Tg mice transplanted with fresh second donor hearts; tolerant IBN-Tg mice accepted donor skin, but rejected F1 skin grafts that expressed donor Ags. We cannot exclude that tolerant IBN-Tg mice have developed a low level of regulation that we are unable to detect with our assays. For instance, although a 1:1 ratio of tolerant to wild-type fresh splenocytes did not prevent wild-type splenocyte-mediated cardiac allograft rejection in RAG1-KO mice, it is possible that a greater ratio would be successful. Nevertheless, we conclude that if regulation exists in this model, it is not dominant, not easily transferable, and of lower magnitude than that induced in B6 mice by anti-CD40L + DST treatment.
In contrast with the lack of evidence for dominant suppression, it was clear that Bcl-xL/IBN-Tg mice were capable of rejecting cardiac allografts. Although NF-B promotes the transcription of multiple antiapoptotic genes, including Bcl-xL, overexpression of just Bcl-xL has been shown to be sufficient to reduce the death of IBN-Tg T cells (23). However, it is also possible that transgenic expression of Bcl-xL in T cells modifies aspects of T cell biology other than survival, and that cardiac allograft rejection in Bcl-xL/IBN-Tg mice is not due to prevention of death of IBN-Tg T cells, but, rather, to increased T cell effector function, for instance. Similar to our model, overexpression of Bcl-xL in T cells has previously been reported to prevent tolerance induction in mice treated with a combination of donor-specific transfusion with CTLA-4-Ig or anti-CD154 (22). These results support the idea that reducing T cell death can prevent transplantation tolerance in models in which apoptosis is the major mechanism of tolerance, although in neither case can it be excluded that Bcl-xL is operating through alternative mechanisms than prevention of apoptosis. Future experiments using TCR transgenic/IBN-Tg mice to be able to follow the fate of alloreactive T cells after transplantation will help determine whether the effect of Bcl-xL depends on prevention of T cell death.
The importance of the precursor frequency of graft-specific T cells in the success or failure to reject a transplant is becoming increasingly apparent (51, 52). Because regulation of alloresponses can coexist with a certain level of deletion of alloreactive T cells, it has been suggested that it may be possible to tip the balance of these two mechanisms in favor of transplantation tolerance (53). In fact, in the absence of regulation or constant depletion of new emerging T cells, T cell deletion may impede transplantation tolerance as new developing T cells may undergo homeostatic expansion to reconstitute the T cell repertoire and thus acquire memory characteristics that confer resistance to certain regimens of tolerance induction, such as costimulatory blockade (54). It is possible that IBN-Tg mice remain tolerant indefinitely without histological evidence of chronic graft rejection (14) because of the continuous deletion of new emerging alloreactive T cells as they encounter alloantigen in the presence of blunted NF-B activation.
The pathway that leads to NF-B activation in T cells downstream of TCR/CD28 engagement is unique. It involves recruitment and activation of protein kinase C, the adaptors CARMA1, Bcl10, and MALT1 and the recruitment of a ubiquitinating complex that includes TRAF6 and results in activation of TAK1, followed by activation of the IKK complex (55). Protein kinase C is mostly restricted to T cells, whereas CARMA1, Bcl10, and MALT1 appear lymphocyte-restricted. Therefore, it may be possible to develop small molecule inhibitors that are cell-permeable and inhibit the binding or activation of these lymphocyte-restricted proteins. These therapies should only inhibit TCR- or BCR-mediated NF-B activation and may reproduce in normal mice or patients the susceptibility to tolerance observed in IBN-Tg mice. However, whether transient deletion of alloreactive T cells will be sufficient to induce and maintain tolerance and whether these therapies will be effective to induce deletion of pre-existing cross-reactive memory cells remain to be established.
Disclosures
The authors have no financial conflict of interest.
Acknowledgments
We thank James Marvin and Ryan Duggan for expert help with cell sorting.
Footnotes
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 This work was supported by American Heart Association Grant 0350261N, Juvenile Diabetes Research Foundation Grant 1-2003-188, and National Institutes of Health Grant RO1AI052352-01.
2 P.Z. and S.J.B. contributed equally to this work.
3 Current address: Department of Immunology, College of Pharmacy, Chuang Ang University, Seoul 156756, Korea.
4 Address correspondence and reprint requests to Dr. Maria-Luisa Alegre, University of Chicago, 5841 S. Maryland Avenue, Room N005C, MC 0930, Chicago, IL 60637. E-mail address: malegre{at}midway.uchicago.edu
5 Abbreviations used in this paper: RHD, Rel homology domain; DST, donor-specific transfusion; IKK, IB kinase; RAG1-KO, RAG1-deficient; Treg, regulatory T cell.
Received for publication June 7, 2004. Accepted for publication January 9, 2005.
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